Pharmaceutical composition for the treatment of autism

ABSTRACT

Likewise, the present invention relates to methods of treating patients suffering from autism spectrum disorder (ASD) phenotype 1 by administering an effective amount of a substance capable of raising intracellular cAMP levels and, optionally, an effective amount of a substance capable of modulating intracellular calcium concentration.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application claims priority from EP 17200219.8, filed Nov. 6, 2017,EP 18169952.1, filed Apr. 27, 2018, Provisional Application 62/582,141,filed Nov. 6, 2017, and Provisional Application 62/663,647, filed Apr.27, 2018, all of which are incorporated by reference in their entirety.

FIELD OF THE INVENTION

The invention relates to a pharmaceutical composition for treating thecore symptoms of autism spectrum disorder (ASD) and/or associatedsymptoms including learning disabilities, language impairment andimpairments in executive functioning that can be associated with thedisease in patients with ASD—and preferably in phenotype 1 patients—asubcategory of patients presenting with specific differentiatingclinical sets of signs and symptoms.

BACKGROUND OF THE INVENTION

Autism spectrum disorder (ASD) are a group of neurodevelopmentaldisorders frequently characterized by impairments in socialinteractions, difficulties with language and communication, and thepresence of repetitive, perseverative behaviors. ASD typically appearsduring the first three years of life and manifests in characteristicsymptoms or behavioral traits. A diagnosis of ASD currently includesseveral conditions that used to be diagnosed separately: autisticdisorder, pervasive developmental disorder not otherwise specified(PDD-NOS), and Asperger syndrome. All of these conditions are nowencompassed by the diagnostic criteria for autism spectrum disorder asset forth in the American Psychiatric Association's Diagnostic &Statistical Manual of Mental Disorders, Fifth Edition (DSM-5).

While ASD is currently defined by symptoms in core areas, there existssignificant heterogeneity in genetics, phenotypes, clinicalpresentation, and associated comorbidities The genetic contribution tothe causation/predisposition to autism is considered to be substantialon the basis of high concordance in monozygous twins s contribute 83% ofthe risk for ASD, and environmental factors thus seem to play a minor17% though significant role in the developmental etiology of ASD.However, to further complicate matters, genetic and epigenetic factorsintertwine with prenatal and lifelong dynamic environmental factors toinfluence individual patient pathogenesis. Nevertheless, causal geneticfactors can only be identified in 15 to 20% of patients, and the vastmajority ASD patients are still considered idiopathic.

A subgroup of ASD patients, the so called ASD phenotype 1 (see EP17200185.1), shows an upregulation of pathways involved in adaptation tostress, apoptosis or cell differentiation, cell proliferation, cellcycle progression, cell division, and differentiation. In particular,but not limited to, the PI3K, AKT, mTOR, MAPK, ERK/JNK-P38 pathways havebeen implicated in autism. Patients diagnosed with ASD phenotype 1 alsoexhibit a significant increase in proinflammatory cytokines (TNF-α,IL-6, IL-1β, IL-17A, IL-22 and GM-CSF), Th1 cytokine (INF-γ) andchemokine (IL-8) expression in the brain compared to healthy controlpatients, whereas no significant differences between ASD phenotype 1patients and normal controls were shown for the Th2 cytokines (IL-4,IL-5) and IL-10. Accordingly, the Th1/Th2 ratio may be significantlyincreased in the ASD Phenotype 1 patients.

Currently, there is no effective treatment for the ASD Phenotype 1subgroup of patients. In fact, these patients react negatively toadministration of antioxidant substances, despite the fact that thesehave been reported to improve some patients with autism.

Fragile X is the most common monogenetic cause of both intellectualdisability and autism spectrum disorder. Patients with Fragile Xsyndrome (FXS), caused by loss of function of the fragile X mentalretardation 1 (Fmr1) gene, often exhibit many of the symptoms commonlyassociated with ASD, such as developmental delays, communicationimpairments and anxiety.

Despite these studies of neurodevelopmental disorders such as ASD, ASDPhenotype 1, and Fragile X syndrome, there remains a need for noveltreatments of neurodevelopmental disorders such as ASD, ASD phenotype 1,and Fragile X Syndrome.

SUMMARY OF THE INVENTION

The present invention meets this need by providing methods andpharmaceutical compositions or kits for treating patients diagnosed withan autism spectrum disorder (ASD), ASD phenotype 1, or fragile Xsyndrome.

In one aspect of the present disclosure, the pharmaceutical compositioncomprises

-   -   a first substance capable of raising intracellular cAMP levels,        and    -   a second substance capable of modulating intracellular calcium        concentration.

In some embodiments, the substance capable of raising intracellular cAMPlevels is a PDE inhibitor. In some embodiments, the PDE inhibitor is aPDE4 inhibitor or a PDE4-3 dual inhibitor. In some embodiments, the PDEinhibitor is selected from the group consisting of theophylline,enprofylline, pentoxifylline, dyphylline, caffeine, dipyridamole,roflumilast, crisaborole, apremilast, cilomilast, tetomilast, rolipram,(S)-rolipram, (R)-rolipram, amrinone, milrinone, enoximone, daxalipram(R-mesopram), lirimilast, AWD-12-281, cipamfylline, oglemilast,tofimilast, CI-1044, HT-0712, MK-0873, arofylline, CI-1018, T-2585,YM-976, V-11294A, piclamilast, atizoram, filaminast, SCH 351591, IC-485,D-4418, CDP-840, and L-826,141. In some embodiments, the substancecapable of raising intracellular cAMP levels comprises a substancecapable of activating a G protein-coupled adenosine A_(2A) receptor. Insome embodiments, the substances capable of activating a Gprotein-coupled adenosine A_(2A) receptor is selected from the groupconsisting of regadenoson, bidenoson, adenosine, 2-phenilaminoadenosine,2-amino-4-(4-hydroxyphenyl)-6-[(1H-imidazol-2-ylmethyl)thio]-3,5-pyridinecarbonitrile,4-[2-[(6-amino-9-b-D-ribofuranosyl-9H-purin-2-yl)thio]ethyl]benzenesulfonicacid ammonium salt, 2-hexynyl-5′-N-ethylcarboxamidoadenosine, CGS-21680,and UK-432,097.

In some embodiments, the substance capable of modulating intracellularcalcium concentration comprises a retinoic acid-related orphanreceptor-alpha (RORA) agonist. In some embodiments, the substancecapable of modulating intracellular calcium concentration comprises acalcium channel inhibitor or an inhibitor of the solute carrier family12 member 1, solute carrier family 12 member 1, 2, 4 or solute carrierfamily 12 member 1, 2, 4, 5. In some embodiments, the substance capableof modulating intracellular calcium concentration is selected from thegroup consisting of dihydropyridines, phenylakylamines,benzothiazepines, indolazines, aminoglycosides, and 4-substitutedderivatives of sulfamoylbenzoic acid. In some embodiments, the4-substituted derivative of sulfamoylbenzoic acid is a derivative of4-substituted-3-amino-5-sulfamoylbenzoic acid.

In some embodiments, the derivative of4-substituted-3-amino-5-sulfamoylbenzoic acid is selected from the groupconsisting of bumetanide, AqB007, AqB011, PF-2178, BUM13, BUM5,bumepamine, and mixtures thereof.

In some embodiments, the substance capable of modulating intracellularcalcium concentration is selected from the group consisting ofnifedipine, niludipine, nicardipine, nimodipine, NZ-105, amlodipine,felodipine, isradipine, diperdipine, emopamil, devapamil, verapamil,diltiazem, flunarizine, fluspirilene, pimozide, fantofarone,nicergoline, neomycin, gentamycin, kanamycin, cisapride, clopamide,cyproheptadine, loratadine, domperidone, fentanyl, alfentanil,sufentanil, flecainide, indoramin, isonepecotic acids, ketotifen,loperamide, mepivacaine, methylphenidate, minoxidil, nipecotic acid,paroxetine, pempidine, penfluridol, perhexiline, pipecolics acid,bupivacaine, cyclohexemide, thalidomide, terfenadine, trihexyphenidyl,clevidipine, lomerazine, fostedil, anipamil, torasemide, chlorthalidone,etacrynic acid, furosemide, trichlormethiazide, hydroflumethiazide,methylclothiazide, bumetanide, ibutilde, mibefradil, dronedarone,amiodarone, nisoldipine, nitrendipine, nilvadipine, gabapentine, andambroxol hydrochloride.

In some embodiments, the substance capable of raising intracellular cAMPlevels is ibudilast, and the substance capable of modulatingintracellular calcium concentration is bumetanide. In some embodiments,A method of treating a patient diagnosed with Fragile X syndromecomprising administering to a patient in need thereof an effectiveamount of a pharmaceutical composition comprising a first substancecapable of raising intracellular cAMP levels and an effective amount ofa second substance capable of modulating intracellular calciumconcentration.

In accordance with some embodiments, there are provided methods oftreating a patient diagnosed with autism spectrum disorder (ASD)comprising administering to a patient in need thereof an effectiveamount of a pharmaceutical composition comprising a first substancecapable of raising intracellular cAMP levels, and a second substancecapable of modulating intracellular calcium concentration.

In another aspect, there are provided kits comprising

-   -   a first substance capable of raising intracellular cAMP levels,        and    -   a second substance capable of modulating intracellular calcium        concentration.

In some embodiments of the kit, the substance capable of raisingintracellular cAMP levels is a PDE inhibitor. In some embodiments of thekit, the PDE inhibitor is a PDE4 inhibitor or a PDE4-3 dual inhibitor.In some embodiments of the kit, the PDE inhibitor is selected from thegroup consisting of caffeine, theophylline, enprofylline,pentoxifylline, dyphylline, theobromine, aminophylline, prepentofylline,L-reuteri, dipyridamole, cilostazol, etazolate, roflumilast, crisaboroleresembrenone, drotaverin, ibudilast, apremilast, cilomilast, tetomilast,rolipram, (S)-rolipram, (R)-rolipram, amrinone, milrinone, enoximone,daxalipram (R-mesopram), lirimilast, AWD-12-281, cipamfylline,oglemilast, tofimilast, CI-1044, HT-0712, MK-0873, arofylline, CI-1018,T-2585, YM-976, V-11294A, piclamilast, atizoram, filaminast, SCH 351591,IC-485, D-4418, CDP-840, and L-826,14.

In some embodiments of the kit, the substance capable of raisingintracellular cAMP levels is a substance capable of activating a Gprotein-coupled adenosine A_(2A) receptor. In some embodiments of thekit, the substance capable of activating a G proteincoupled adenosineA_(2A) receptor is selected from the group consisting of regadenoson,bidenoson, adenosine, 2-phenilaminoadenosine,2-amino-4-(4-hydroxyphenyl)-6-[(1H)-imidazol-2-ylmethyl)thio]-3,5-pyridinecarbonitrile,4-[2-[(6-amino-9-b-D-ribofuranosyl-9H-purin-2-yl)thio]ethyl]benzenesulfonicacid ammonium salt and 2-hexynyl-5′-N-ethylcarboxamidoadenosine,CGS-21680 and UK-432,097. In some embodiments of the kit, the substancecapable of modulating intracellular calcium concentration is a retinoicacid-related orphan receptor-alpha (RORA) agonist

In some embodiments of the kit, the substance capable of modulatingintracellular calcium concentration is a calcium channel inhibitor or aninhibitor of the solute carrier family 12 member 1, an inhibitor of thesolute carrier family 12 member 1, 2, 4 or an inhibitor of the solutecarrier family 12 member 1, 2, 4, 5.

In some embodiments of the kit, the substance capable of modulatingintracellular calcium concentration is selected from the groupconsisting of dihydropyridines, phenylakylamines, benzothiazepines,indolazines, aminoglycosides and a 4-substituted derivative ofsulfamoylbenzoic acid. In some embodiments of the kit, the 4-substitutedderivative of sulfamoylbenzoic acid is selected from the groupconsisting of bumetanide, AqB007, AqB11, PF-2178, BUM13, BUM5,bumepamine and mixtures thereof. In some embodiments of the kit, the4-substituted derivative of sulfamoylbenzoic acid is a derivative of4-substituted-3-amino-5-sulfamoylbenzoic acid.

In some embodiments of the kit, the substance that modulatesintracellular calcium concentration is selected from the groupconsisting of nifedipine, niludipine, nicardipine, nimodipine, NZ-105,amlodipine, felodipine, isradipine, diperdipine, emopamil, devapamil,verapamil, diltiazem, flunarizine, fluspirilene, pimozide, fantofarone,nicergoline, neomycin, gentamycin, kanamycin, cisapride, clopamide,cyproheptadine, loratadine, domperidone, fentanyl, alfentanil,sufentanil, flecainide, indoramin, isonepecotic acids, ketotifen,lobeline, loperamide, mepivacaine, methylphenidate, minoxidil, nipecoticacid, paroxetine, pempidine, penfluridol, perhexiline, pipecolics acid,bupivacaine, cyclohexemide, thalidomide, terfenadine, trihexyphenidyl,clevidipine, lomerazine, fostedil, anipamil, torasemide, chlorthalidone,etacrynic acid, furosemide, trichlormethiazide, hydroflumethiazide,methylclothiazide, bumetanide, ibutilde, mibefradil, dronedarone,amiodarone, nisoldipine, nitrendipine, nilvadipine, gabapentine andambroxol hydrochloride.

In some embodiments of the kit, the substance capable of raisingintracellular cAMP levels comprises ibudilast and the substance capableof modulating intracellular calcium concentration comprises bumetamide.

In some embodiments, the kit disclosed herein is for use in thetreatment of autism spectrum disorder (ASD). In some embodiments, thekit disclosed herein is for use in the treatment of Fragile X syndrome

In another aspect, the methods disclosed herein is for treating patientexhibits characteristics consistent with being an ASD phenotype 1patient. In some embodiments of the method for treating an ASD phenotype1 patient, the substance capable of raising intracellular cAMP levels isa PDE inhibitor. In some embodiments of the method for treating an ASDphenotype 1 patient, the PDE inhibitor is a PDE4 inhibitor or a PDE4-3dual inhibitor. In some embodiments of the method for treating an ASDphenotype 1 patient wherein the PDE inhibitor is ibudilast. In someembodiments of the method for treating an ASD phenotype 1 patient, thepharmaceutical composition is administered to the patient at a dailytotal dosage ranging from about 1-150 mg, preferably from about 10-80mg, and more preferably from about 15-50 mg, divided among one, two, orthree doses. In some embodiments of the method for treating an ASDphenotype 1 patient, the pharmaceutical composition is administeredorally once, twice, or thrice daily to the patient using a dosage formthat comprises 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 mg ibudilast,or a pharmaceutically acceptable salt thereof.

In another aspect, there are provided methods of treating a patientdiagnosed with autism spectrum disorder (ASD), wherein the treatmentcomprises

-   -   (i) determining whether the patient suffers from ASD phenotype        1, and    -   (ii) administering a therapeutically effective amount of a        pharmaceutical composition comprising a first substance capable        of raising intracellular cAMP levels, and a second substance        capable of modulating intracellular calcium concentration to the        patient if the patient suffers from ASD phenotype 1;

wherein determining whether the patient suffers from ASD phenotype 1includes at least one step selected from

-   -   a) subjecting the patient to a challenge test with        Nrf2-activator,    -   b) verifying for clinical signs of heightened expression of        proliferation-associated pathways,    -   c) verifying for upregulation of Nrf2, or    -   d) verifying for low levels of protein kinase A; and

wherein it is determined that the patient suffers from ASD phenotype 1if the patient exhibits at least one characteristic selected fromnegative behavioral response in the challenge test with a Nrf2 inducer,clinical signs of heightened expression of proliferation-associatedpathways, upregulation of Nrf2, and low blood levels of protein kinaseA.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure provides novel methods and compositions fortreating patients diagnosed with autism spectrum disorder (ASD), inparticular subtype ASD phenotype 1, and Fragile X syndrome. The hereindisclosed methods, compositions and kits are based on the surprisingdiscovery, illustrated in the Example herein, that the effect ofsubstances capable of increasing intracellular cyclic adenosinemonophosphate (cAMP) on treating cognitive dysfunctions associated withASD can be synergistically and unexpectedly improved by co-treatmentwith substances capable of modulating intracellular calciumconcentration. Accordingly, in one aspect, the present invention relatesto a pharmaceutical composition or a kit comprising

a) a first substance capable of raising intracellular cAMP levels, and

b) a second substance capable of modulating intracellular calciumconcentration.

The present disclosure is based on the surprising discovery, exemplifiedin the example herein, that ibudilast, a substance capable of raisingintracellular cAMP levels, and bumetamide, a substance capable ofmodulating intracellular calcium concentration, in combination can treatpatients diagnosed with ASD phenotype 1.

I. Definitions

Technical and scientific terms used herein have the meanings commonlyunderstood by one of ordinary skill in the art to which the presentinvention pertains, unless otherwise defined. Materials, reagents andthe like to which reference is made in the following description andexamples are obtainable from commercial sources, unless otherwise noted.

As used herein, the singular forms “a,” “an,” and “the” designate boththe singular and the plural, unless expressly stated to designate thesingular only.

The term “about” means that the number comprehended is not limited tothe exact number set forth herein, and is intended to refer to numberssubstantially around the recited number while not departing from thescope of the invention. As used herein, “about” will be understood bypersons of ordinary skill in the art and will vary to some extent on thecontext in which it is used. If there are uses of the term which are notclear to persons of ordinary skill in the art given the context in whichit is used, “about” will mean up to plus or minus 10% of the particularterm.

A “kit” is herein defined as combination product provided as a packageand containing several individual parts that show a complementary effectwhen applied together. In this aspect, the effect achieved by a kit anda pharmaceutical composition are similar. A kit offers the advantagethat dosage regimens of the individual parts may be adjusted to specificrequirements arid over time.

As used herein “subject,” “patient,” or “individual” refers to anysubject, patient, or individual, and the terms are used interchangeablyherein. When used in conjunction with “in need thereof,” the term“subject,” “patient,” or “individual” intends any subject, patient, orindividual having or at risk for a specified symptom or disorder.

As used herein, the term “administering” includes directly administeringto another, self-administering, and prescribing or directing theadministration of an agent as disclosed herein.

As used herein, the phrases “effective amount” and “therapeuticallyeffective amount” mean that active agent dosage or plasma concentrationin a subject, respectively, that provides the specific pharmacologicaleffect for which the active agent is administered in a subject in needof such treatment. It is emphasized that an effective amount of anactive agent will not always be effective in treating theconditions/diseases described herein, even though such dosage is deemedto be an effective amount by those of skill in the art.

As used herein, the term “pharmaceutical composition” refers to one ormore active agents formulated with a pharmaceutically acceptablecarrier, excipient or diluent.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in vivowithout excessive toxicity, irritation, allergic response, or otherproblem or complication, commensurate with a reasonable benefit/riskratio.

II. Autism Spectrum Disorders

As used herein, the term “autism spectrum disorder (ASD)” is understoodto cover a family of neurodevelopmental disorders characterized bydeficits in social communication and interaction and restricted,repetitive patterns of behavior, interests or activities. In thefollowing, the terms “autism spectrum disorder”, “autism” and “ASD” areused interchangeably.

Herein, the terms “ASD phenotype 1” and “phenotype 1” are usedinterchangeably.

The term “ASD patient” is intended to cover not only humans diagnosed ashaving ASD, but also humans suspected of having ASD.

The person skilled in the art is well aware of how a patient may bediagnosed with ASD. For example, the skilled person may follow thecriteria set up in “American Psychiatric Association; Diagnostic andStatistical Manual of Mental Disorders (DSM-5) Fifth edition” to give asubject a diagnosis of ASD. Likewise, ASD patients may have beendiagnosed according to standardized assessments tools including but notlimited to CIM-10, ICD-10, DISCO, ADI-R, ADOS or CHAT.

In other cases, patients may have a well-established DSM-IV diagnosis ofautistic disorder, Asperger's disorder, or pervasive developmentaldisorder not otherwise specified (PDD-NOS).

Additionally, the present invention may be useful for subjectsfulfilling one or more of the following criteria: persistent deficits insocial communication and social interaction across multiple contexts asmanifested by the following, currently or by history; restricted,repetitive patterns of behavior, interests, or activities, as manifestedby at least two of the following, currently or by history; symptomspresent in the early development period (but may not become fullymanifest until social demands exceed limited capacities, or maybe maskedby learned strategies in later life); symptoms cause clinicallysignificant impairment in social, occupational, or other important areasof current functioning; these disturbances are not better explained byintellectual disability (intellectual development disorder) or globaldevelopment delay.

ASD may occur with or without accompanying intellectual and/or languageimpairment. It may be associated with a known medical or geneticcondition or an environmental factor or other neurodevelopmental, mentalor behavioral disorders.

ASD may occur in different severity levels which may be classifiedaccording to impairment in social communication and in terms ofrestricted, repetitive behavior. Importantly, the term ASD phenotype 1is not associated with a particular severity level of ASD. The presentinvention may be applied to patients suffering from any severity levelof ASD.

Without being bound by a mechanism, it is believed that ASD patients canbe characterized depending on whether or not they show an upregulation,a downregulation or normal levels of expression of biomolecular pathwaysinvolved in stress response.

Depending on whether and how the level of expression of these pathwaysin the respective individuals are modified, challenging ASD patientswith Nrf2-activators which are known to upregulate the respectivepathways will improve or worsen ASD symptoms.

In one aspect, the pharmaceutical composition or kit according to thepresent invention is for use in the treatment of an ASD or of a subgroupof ASD patients called ASD phenotype 1 patients.

ASD phenotype 1 patients may be identified with the help of a challengetest as described in non-published EP17200185.1, incorporated herein byreference in its entirety. Briefly, the concept of a challenge test isbased on administration of an Nrf2-activator to an ASD patient. In ASDphenotype 1 patients, who already show an upregulation of the respectivepathways, further activation of Nrf2 will lead to a worsening of coresymptoms. Consequently, ASD phenotype 1 patients may be identified by anegative behavioral response to a challenge test.

Likewise, the skilled person can identify ASD phenotype 1 patientaccording to the clinical signs as defined in EP 17200185.1.

Another way of identifying ASD phenotype 1 patient is to check forupregulation of Nrf2. The person skilled in the art is well aware of howupregulation of the expression of a specific gene such as Nrf2 may beinvestigated. For example, it may be investigated at the mRNA levelusing quantitative PRC techniques such as qPCR or RT-qPCR. Likewise,upregulation of the expression of a gene may be determined on theprotein level using protein quantification techniques such as WesternBlot and quantitative dot blots. Upregulation is understood to mean anincrease of mRNA levels or protein levels of at least 10% when comparedto samples from healthy subjects.

Additionally, an ASD phenotype 1 patient may exhibit one or more of thefollowing symptoms: increased levels of TH1 and pro-inflammatory seracytokines (including but not limited to TNF-α, IL-1β, IL-6, Il-17A andIL-22) and/or of tissue expressions of mRNAs that encode inflammatorycytokines and/or excessive challenged T cell secretion ofproinflammatory cytokines; upregulation of pathways involved inadaptation to stress, apoptosis, cell differentiation, cellproliferation, cell cycle progression, cell division and differentiationparticular but not limited to PI3K, AKT, mTOR/MAPK, ERK/JNK-P38).

Currently, there remain a need for effective treatment available forpatients diagnosed with autism spectrum disorders (ASDs), and inparticular ASD phenotype 1. The present disclosure meets this need byproviding a novel method of treating ASD and ASD phenotype 1 inparticular by administering substances capable of raising intracellularcAMP levels and substances capable of modulating intracellular calciumconcentration, which is unprecedented in the literature.

III. Pharmaceutical Compositions and Methods for Treatment of Autism.

The methods and compositions described herein are based on thesurprising discovery that the effect of treating patients diagnosed withan autism spectrum disorder with substances capable of raisingintracellular cyclic Adenosine Monophosphate (cAMP) levels can besynergistically and unexpectedly enhanced by co-treatment withsubstances capable of modulating intracellular calcium levels. Thiscombined treatment with substances capable of raising intracellular cAMPand substances capable of modulating intracellular calcium levels isunprecedented in literature, and provides a novel method of treatingautism spectrum disorders and fragile X syndrome.

a. Substances Capable of Raising Intracellular cAMP Levels

Cyclic Adenosine Monophosphate (cAMP) is synthetized from adenosinetriphosphate (ATP) via the action of adenylyl cyclase (AC) and isconverted to its inactive form 5′-adenosin monophosphate (5′-AMP) viahydrolysis by phosphodiesterase (PDE). Accordingly, one class ofsubstances available for increasing cAMP levels is inhibitors of PDE.

In one aspect, the substance capable of increasing the intracellularlevels of cAMP is a phosphodiesterase (PDE) inhibitor. Sincephosphodiesterases convert cAMP to its inactive form, inhibitingphosphodiesterases will increase the levels of cAMP. One type ofcAMP-PDEs is the PDE4 family which comprises four genes, PDE4A to D.PDE4s differ from other cAMP-PDEs by their kinetic properties and,particularly, their sensitivity to inhibition by the prototypical PDE4inhibitor rolipram.

Accordingly, the present disclosure provides pharmaceutical compositionsthat comprises a substance that is capable of raising intracellular cAMPlevels. The substance may be capable of raising intracellular cAMPlevels directly or indirectly or both. A substance capable of directlyraising intracellular cAMP levels may directly stabilize cAMP or inhibitdegradation of cAMP. A substance capable of indirectly raisingintracellular cAMP levels may activate molecules which lead to thegeneration of cAMP or may inhibit molecules degrading cAMP.

Inhibition is herein understood to mean blocking or reducing theactivity of the respective molecule. In some embodiments, activity maybe reduced by more than 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%. In apreferred embodiment, activity is reduced by at least 50%.

Activation is herein understood to mean upregulatin.g or enhancing theactivity of the respective molecule. In some embodiments, activity maybe enhanced by more than 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%. In apreferred embodiment, activity may be enhanced by at least 50%.

Substances capable of inhibition of phosphodiesterase may be anymolecule having a c-AMP-specific 3′-5′-cyclic phosphodiesterase 4A/B/Cinhibitory effect, any molecule having a c-AMP 3′-5′-cyclicphosphodiesterase 4A/B inhibitory effect, any molecule having a cAMP3′-5′ phosphodiesterase 4A inhibitory effect, any molecule having a cAMP3′-5′-cyclic phosphodiesterase 4A/B/C/D inhibitory effect, any moleculehaving a cAMP 3′-5′-cyclic phosphodiesterase 4A/B/D inhibitory effect,any molecule having a cAMP 3′-5′ phosphodiesterase 4/B/D inhibitoryeffect, any molecule having a cAMP 3′-5′-cyclic phosphodiesterase 4Dinhibitory effect, any molecule having a cAMP 3′-5′-cyclicphosphodiesterase 4B/D inhibitory effect, any molecule having a cAMP3′-5′-cyclic phosphodiesterase 4B inhibitory effect.

Substances capable of inhibition of PDE include pyrazopyridines,preferably ibudilast, xanthines and derivatives, in particularenprofylline, pentoxifylline, dyphylline, aminophylline,propentofylline, preferably caffeine, theobromine, theophilline;flavonoids, in particular xanthohumol, leueocyanidol, delphinidin;flavones, in particular apigenin, luteolin; biflavones, in particularpodocarpusflavone A; sequoiaflavones, in particular podocarpusflacvoneB, 7,7″-dio-methylaminoflavone, bilobetin; flavanones, in particulardioclein, naringenin, hesperetin; stilbens, in particularE-(epsilon)-viniferin, curcumin; alkaloids, in particular chelerythrine,glaucine, apomorphine; aminopyrimidines and derivatives, in particulardiakylarylamines, preferably dipyridamole; arenecarboxamides inparticular benzamides, preferably roflumilast or piclamilast;benzoxaboroles, in particular crisaborole; isoindolones, in particularphthalimides, preferably apremilast; methoxybenzenes, in particularcilomilast; pyridinecarboxylic acids, in particular tetomilast;phenylpyrrolidines, in particular pyrrolidin-2-ones, preferablyrolipram, (S)-rolipram, (R)-rolipram; bipyridines; oligopyridines, inparticular amrinone, milrinone; arylphenylketones, in particularenoximone; oxazolidin-2-ones, in particular daxalipram (R-mesopram);probiotics, in particular L-reuteri.

In a preferred embodiment, the substance capable of inhibiting PDE isselected from the group consisting of ibudilast, caffeine, theobromine,theophylline, enprofylline, pentoxifylline, dyphylline,L-reuteri,dipyridamole, cilostazol, etazolate, roflumilast, crisaboroleresembrenone, drotaverin, apremilast, cilomilast, tetomilast, rolipram,(S)-rolipram, (R)-rolipram, amrinone, milrinone, enoximone, daxalipram(R-mesopram), lirimilast, AWD-12-281, cipamfylline, oglemilast,tofimilast, CI-1044((R)—N-(9-amino-4-oxo-1-phenyl-3,4,6,7-tetrahydro-[1,4]diazepino[6,7,1-hi]indol-3-yl)nicotinamide),HT-0712 ((3S,5S)-2-Piperidinone,5-(3-(cyclopentyloxy)-4-methoxyphenyl)-3-((3-methylphenyl)methyl),MK-0873(3-(2-{3-[3-(cyclopropylcarbamoyl)-4-oxo-1,4-dihydro-1,8-naphthyridin-1-yl]phenyl}ethynyl)pyridin-1-ium-1-olate),arofylline, CI-1018(N-(3,4,6,7-tetrahydro-9-methyl-4-oxo-1-phenylpyrrolo(3,2,1-jk)(1,4)benzodiazepin-3-yl)-4-Pyridinecarboxamide),T-2585(2-{4-2,3-bis(hydroxymethyl)-6,7-diethoxy-1-naphthalenyl}-2-pyridinyl]-4-(3-pyridinyl)-1(2H)-phthalazinone),YM-976(4-(3-chlorophenyl)-1,7-diethyl-1H,2H-pyrido[2,3-d]pyrimidin-2-one),V-11294A (3-(3-Cyclopentyloxy-4-methoxy-benzyl)-8-isopropyl-adenine),piclamilast, atizoram, filaminast, SCH 351591(N-(3,5-Dichloro-1-oxido-4-pyridinyl)-8-methoxy-2-(trifluoromethyl)-5-quinolinecarboxamide),IC-485, D-4418(N-(2,5-dichloro-3-pyridinyl)-8-methoxy-5-quinolinecarboxamide), CDP-840(4-[(2R)-2-[3-(Cyclopentyloxy)-4-methoxyphenyl]-2-phenylethyl]-pyridinehydrochloride), L-826,141(4-(2-(3,4-bis(difluoromethoxy)phenyl)-2-(4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)phenyl)ethyl)-3-methylpyridine1-oxide), BPN14770(2-(4-((2-(3-chlorophenyl)-6-(trifluoromethyl)pyrimidin-4-yl)amino)phenyl)acetamide),TDP101.

More preferred, the substance capable of inhibiting PDE is ibudilast.

Ibudilast is an anti-inflammatory and neuroprotective oral agent,metabolized mainly by the liver. Following administration of a singledose of 10 mg ibudilast to healthy adults, about 60% of the dose wasexcreted as metabolites in urine in 72 hours. The clinical efficacy ofthis product has been proven for bronchial asthma indication andcerebrovascular disorders. Ibudilast is currently under clinical trialin the U.S. for progressive multiple sclerosis and other conditions suchas amyotrophic lateral sclerosis and substances dependence (codes:AV-411 or MN-166).

Substances that are capable of indirectly raising intracellular cAMPlevels include substances capable of stimulating a G protein-coupledadenosine A_(a2) receptor which activates adenylate cyclase, substancescapable of activation of adenylate cyclase, substances that are capableof inhibiting glycogen synthetase kinase-3-beta (GSK-3-beta), as well assubstances capable of inhibition of phosphodiesterase.

More specifically, a substance capable of activation of G protein-coupleadenosine A_(a2) receptor may be a purine nucleoside, in particularregadenoson, bidenoson, adenosine, 2-phenilaminoadenosine,2-amino-4-(4-hydroxyphenyl)-6-[(1H-imidazol-2-ylmethyl)thio]-3,5-pyridinecarbonitrile,4-[2-[(6-amino-9-b-D-ribofuranosyl-9H-purin-2-yl)thio]ethyl]benzenesulfonicacid ammonium salt, 2-hexynyl-5′-N-ethylcarboxamidoadenosine, CGS-21680or UK-432,097.

Substances capable of inhibiting glycogen synthetase kinase-3-beta(GSK-3-beta) may be lithium salts.

b. Substances Capable of Modulating Intracellular Calcium Levels

The pharmaceutical composition according to the present invention mayoptionally comprise a substance capable of modulating intracellularcalcium levels.

Herein, the term “modulating” may refer to a global decrease ofcytosolic intracellular calcium, a localized increase of intracellularcalcium in specific organelles, such as the mitochondria or endoplasmicreticulum, which in turns lead to a decrease of the cytosolic calciumconcentration, or a modulation of the dynamics of intracellular calcium,such as a modulation of intracellular calcium vague, intracellularcalcium oscillations, and/or intracellular calcium sparks.

The substance capable of modulating intracellular calcium levels maymodulate intracellular calcium levels by mechanisms including eitherinhibition of intracellular calcium channels such as ryanodine receptor(RyR) or inositol-1,4,5-triphosphate-receptor (IP3R), or direct orindirect modulation of calcium-ATPase pumps, including Sarcoendoplasmicreticulum Ca2+ transport-ATPase (SERCA) and Plasma Membrane Ca2+ ATPase(PMCA), ionic exchangers, such as Na+/Ca2+ exchangers, Na+/H+exchangers, Na+/K+ exchangers, NKKC or calcium channels.

The substance capable of modulating intracellular calcium levels may beany molecule having an inhibitory effect on solute carrier family 12member 1, on solute carrier family 12 member 1,2,4 or on solute carrierfamily 12 member 1,2,4, 5.

Likewise, the substance capable of modulating intracellular calciumlevels may be a molecule having an inhibitory effect onvoltage-dependent calcium channels, preferably on voltage-dependentL-type, N-type or T-type calcium channels. In a preferred embodiment,the substance capable of modulating intracellular calcium levels may bea molecule having an inhibitory effect on voltage dependent L-typecalcium channel subunits beta-1/-4, subunit beta-2 or subunitsalpha-1/delta-2. The substance capable of modulating intracellularcalcium levels may be a substance that inhibits the NKCC co-transporter.

In one embodiment, the substance capable of modulating intracellularcalcium levels can be a diuretic. In another embodiment, the substancecapable of modulating intracellular calcium levels can be anantiarrythmic agent. The skilled person is well aware of which drug orpharmaceuticals fall under the terms “diurectic” and “antiarrythmicagent”.

In a preferred embodiment, the substance capable of modulatingintracellular calcium levels may be selected from pyridinesulfonamides,in particular torasemide; isoindolines, in particular chlorthalidone;chlorophenoxyacetates, in particular etacrynic acid;aminobenzenesulfonamides, in particular furosemide and bumetanide;4-substituted-3-amino-5-sulfamoylbenzoic acid derivatives, in particularbut not limited to bumetanide, AqB007, AqB011, bumepamine;1,2,4-benzothiadiazine-1,1-dioxides, in particular trichlormethiazide,hydroflumethiazide and methylclothiazide; dimethoxybenzenes, inparticular ibutilde or verapamil; tetralins, in particular mibefradil;aryl-phenylketones, in particular dronedarone and amiodarone;1,4-dihydropyridine and derivatives, in particular amlodipine,felodipine, diperdipine, nifedipine nimodipine, nisoldipine,nitrendipine, clevidipine, nicardipine and nilvadipine; benzoxadiazoles,preferably dihydropyridinecarboxylic acids and derivatives, inparticular isradipine; diphenylmethylpiperazine and derivatives inparticular flunarizine; heteroarylpiperidine in particular pimozide,domperidone; piperidines in particular cyproheptadine, fentanyl,alfentanil, sufentanil, flecainide, loperamide, methylphenidate,paroxetine, pempidine, perhexiline; henzocycloheptapyridine inparticular loratadine; organic heterotetracyclic compound in particularnicergoline; aminoglycoside in particular, neomycin, gentamycin,kanamycin; piperidinecarboxamide in particular mepivacaine, bupivacaine;triptamines in particular indoramin; sulfonamides in particularclopamide; aminobenzamides in particular cisapride; aminopyrimidine inparticular minoxidil; piperidine alkaloid in particular lobeline;piperidine antibiotic in particular cycloheximide non-proteinogenicamino acids such as gamma amino acids and derivatives, in particulargabapentin; piperidinemonocarboxylic acid in particular nipecotic acid,pipecolics acid; diarylmethane in particular penfluridol;benzothiazepine derivatives in particular diltiazem; phthalamides inparticular thalidomide; diarylmethane in particular terfenadine,lomerazine, aromatic amine in particular ambroxol hydrochloride.

In another preferred embodiment, the substance capable of modulatingintracellular calcium levels is selected from the group consisting ofnifedipine, niludipine, nicardipine, nimodipine, NZ-105, amlodipine,felodipine, isradipine, diperdipine, emopamil, devapamil, verapamil,diltiazem, flunarizine, fluspirilene, pimozide, fantofarone,nicergoline, neomycin, gentamycin, kanamycin, cisapride, clopamide,cyproheptadine, loratadine, domperidone, fentanyl, alfentanil,sufentanil, flecainide, indoramin, isonepecotic acids, ketotifen,lobeline, loperamide, mepivacaine, methylphenidate, minoxidil, nipecoticacid, paroxetine, pempidine, penfluridol, perhexiline, pipecolics acid,bupivacaine, cyclohexemide, thalidomide, terfenadine, trihexyphenidyl,clevidipine, lomerazine, fostedil, anipamil, torasemide, chlorthalidone,etacrynic acid, furosemide, trichlormethiazide, hydroflumethiazide,methylclothiazide, bumetanide, ibutilde, mibefradil, dronedarone,amiodarone, nitrendipine, nilvadipine, gabapentine, ambroxolhydrochloride.

In yet another aspect, the pharmaceutical composition according to thepresent invention may optionally comprise a retinoic acid-related orphanreceptor-alpha (RORA) agonist. RORA is a ligand dependent orphan nuclearhormone receptor that, in combination with co-regulator proteins, servesas a transcriptional regulator. RORA agonists may be phenylnaphthalenes,in particular adaptalene; sesquiterpenoids, in particular all-transacitretin; diterpenoids, in particular alitretinoin or isotretinoin;thiochromanes, in particular tazarotene, retinoid esters, in particularetretinate; retinobenzoic acids, in particular tamibarotene,all-trans-retinoic acid or tretinoin. In a preferred embodiment, theRORA agonist may be selected from the group consisting of adapalene,all-trans retinoic acid, tretinoin, vitamin A, all-trans acitretin,alitretinoin, tazarotene, etretinate, isotretinoin, tamibarotene,LGD-1550, AM580 and Ch 55.

in sharp contrast, administering the pharmaceutical compositionsaccording to the present invention, the effect of a substance capable ofmodulating intracellular calcium concentration such as bumetanide setsin within several days.

It is also envisaged by the present invention that the pharmaceuticalcomposition according to the present invention additionally comprisespharmaceutically acceptable carriers or excipients such as lubricants,disintegrants, antiadherents, binders, preservatives, sorbents orvehicles.

In another aspect, the pharmaceutical composition according to thepresent invention may additionally comprise an agent that inhibits cellproliferation. In another aspect, the present invention relates to apharmaceutical composition for use in the treatment of ASD phenotype 1,the pharmaceutical composition comprising a substance capable of raisingintracellular cAMP levels.

In one embodiment, the substance capable of raising intracellular cAMPlevels is preferably a PDE inhibitor. In a particularly preferredembodiment, the PDE inhibitor is ibudilast. Ibudilast may beadministered to the patient at a daily total dosage ranging from about1-150 mg, preferably from about 5-80 mg, and more preferably from about15-50 mg, divided among one, two, or three doses. It may be administeredorally once, twice, or thrice daily to the patient using a dosage formthat comprises 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 mg ibudilastor a pharmaceutically acceptable salt thereof.

In yet another aspect, the substance capable of modulating Ca2+ is aNKKC1 inhibitor. The substance capable of modulating intracellularcalcium levels may be a substance that inhibits the NKCC co-transporter.NKCC co-transporters have been shown to promote rise in intracellularcalcium either alone or in combination with other ions exchangers, suchas Na/Ca exchanger, Na+/H+ exchanger, and/or Na+/K+ exchanger.

In a particularly preferred embodiment, the NKKC1 inhibitor isbumetanide. Bumetanide may be administered to the patient at a dailytotal dosage ranging from about 0.5 to 10 mg, preferably from 1 to 6 mg,and more preferably from 2 mg to 4 mg, divided into one, two, or threedoses. It may be administered orally once, twice, or thrice daily to thepatient using a dosage form that comprises 0.5, 1, 2 mg bumetanide, or apharmaceutically acceptable salt thereof. Administration of a singledose may enhance patient compliance, while administration of severalsmaller doses ensures constant serum levels.

In yet another aspect, the pharmaceutical composition or kit accordingto the present invention may additionally comprise an agent thatinhibits cell proliferation, such as compounds targeting PI3K, AKT,mTOR, MAPK, ERK/JNK-P38 known to modulate cellular proliferation.

In yet another aspect, the present invention relates to a pharmaceuticalcomposition for use in the treatment of ASD in a patient, comprising asubstance capable of raising intracellular cAMP levels, and a substancecapable of modulating intracellular calcium levels, wherein thetreatment comprises:

-   -   (i) determining whether the patient suffers from ASD phenotype        1, and    -   (ii) administering a therapeutically effective amount of the        pharmaceutical composition to the patient if the patient suffers        from ASD phenotype 1;

wherein determining whether the patient suffers from ASD phenotype 1includes at least one selected from:

-   -   a) subjecting the patient to a challenge test with        Nrf2-activator,    -   b) verifying for clinical signs of heightened expression of        proliferation associated pathways,    -   verifying for upregulation of Nrf2, or    -   verifying for low blood levels of protein kinase A; and

wherein it is determined that the patient suffers from ASD phenotype 1if he shows at least one of the following: negative behavioral responsein a challenge test with a Nrf2 activator such as sulforaphane; clinicalsigns of heightened expression of proliferation-associated pathways;upregulation of Nrf2 or low levels of protein kinase A.

In another aspect, the pharmaceutical composition according to theinvention may be used for the treatment of Fragile X.

In another aspect, a method is disclosed for treating a patientdiagnosed with Fragile X Syndrome comprising administering to a patientin need thereof an effective amount of ibudilast or a pharmaceuticallyacceptable salt thereof.

In another aspect, a method is disclosed for treating a patientdiagnosed with autism spectrum disorder (ASD) comprising administeringto a patient in need thereof an effective amount of ibudilast or apharmaceutically acceptable salt thereof.

In another aspect, a method is disclosed in which the patient exhibitscharacteristics consistent with being an ASD subtype 1 patient.

In another aspect, a method is disclosed in which ibudilast or apharmaceutically acceptable salt is administered to the patient at adaily total dosage ranging from about 1-150 mg, preferably from about10-80 mg, and more preferably from about 15-50 mg, divided among one,two, or three doses.

In another aspect, a method is disclosed in which ibudilast or apharmaceutically acceptable salt thereof is administered orally once,twice, or thrice daily to the patient using a dosage form that comprises1, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 mg ibudilast or apharmaceutically acceptable salt thereof.

IV. Pharmaceutical Composition Carriers and Dosage Forms

In some embodiments, a pharmaceutical composition disclosed hereincomprises one or more pharmaceutically acceptable carriers, such as anaqueous carrier, buffer, and/or diluent.

In some embodiments, a pharmaceutical composition disclosed hereinfurther comprises a simple polyol compound, such as glycerin. Otherexamples of polyol compounds include sugar alcohols. In someembodiments, a pharmaceutical composition disclosed herein comprises anaqueous carrier and glycerin at about a 2:1 ratio.

The pharmaceutical composition may conveniently be presented in unitdosage form and may be prepared by any of the methods well known in theart of pharmacy. An exemplary oral dosage form is a tablet or capsule.An exemplary intranasal dosage form is a liquid or powder nasal spray. Anasal spray is designed to deliver drug to the upper nasal cavity, andcan be a liquid or powder formulation, and in a dosage form such as anaerosol, liquid spray, or powder.

The pharmaceutical composition herein may be combined or coordinatelyadministered with a suitable carrier or vehicle depending on the routeof administration. As used herein, the term “carrier” means apharmaceutically acceptable solid or liquid filler, diluent orencapsulating material. A water-containing liquid carrier can comprisepharmaceutically acceptable additives such as acidifying agents,alkalizing agents, antimicrobial preservatives, antioxidants, bufferingagents, chelating agents, complexing agents, solubilizing agents,humectants, solvents, suspending and/or viscosity-increasing agents,tonicity agents, wetting agents or other biocompatible materials. Atabulation of ingredients listed by the above categories can be found inthe U.S. Pharmacopeia National Formulary, 1857-1859, and (1990).

Some examples of the materials which can serve as pharmaceuticallyacceptable carriers are sugars, such as lactose, glucose and sucrose;starches such as corn starch and potato starch; cellulose and itsderivatives such as sodium carboxymethyl cellulose, ethyl cellulose andcellulose acetate; powdered tragacanth; malt; gelatin; talc; excipientssuch as cocoa butter and suppository waxes; oils such as peanut oil,cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; glycols, such as propylene glycol; polyols such asglycerin, sorbitol, mannitol and polyethylene glycol; esters such asethyl oleate and ethyl laurate; agar; buffering agents such as magnesiumhydroxide and aluminum hydroxide; alginic acid; pyrogen free water;isotonic saline; Ringer's solution, ethyl alcohol and phosphate buffersolutions, as well as other nontoxic compatible substances used inpharmaceutical formulations.

Wetting agents, emulsifiers and lubricants such as sodium lauryl sulfateand magnesium stearate, as well as coloring agents, release agents,coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions,according to the desires of the formulator. Examples of pharmaceuticallyacceptable antioxidants include water soluble antioxidants such asascorbic acid, cysteine hydrochloride, sodium bisulfite, sodiummetabisulfite, sodium sulfite and the like; oil-soluble antioxidantssuch as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylatedhydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol and thelike; and metalchelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid andthe like.

Pharmaceutical compositions according to the invention may also compriseone or more binding agents, filling agents, lubricating agents,suspending agents, sweeteners, flavoring agents, preservatives, buffers,wetting agents, disintegrants, effervescent agents, and otherexcipients. Such excipients are known in the art. Examples of fillingagents include lactose monohydrate, lactose anhydrous, and variousstarches; examples of binding agents include various celluloses andcross-linked polyvinylpyrrolidone, microcrystalline cellulose, such asAvicel® PH101 and Avicel® PH102, microcrystalline cellulose, andsilicified microcrystalline cellulose (ProSolv SMCC™). Suitablelubricants, including agents that act on the flowability of the powderto be compressed, may include colloidal silicon dioxide, such asAerosil® 200, talc, stearic acid, magnesium stearate, calcium stearate,and silica gel. Examples of sweeteners may include any natural orartificial sweetener, such as sucrose, xylitol, sodium saccharin,cyclamate, aspartame, and acesulfanic. Examples of flavoring agents areMagnasweet® (trademark of MAFCO), bubble gum flavor, and fruit flavors,and the like. Examples of preservatives include potassium sorbate,methylparaben, propylparaben, benzoic acid and its salts, other estersof parahydroxybenzoic acid such as butylparaben, alcohols such as ethylor benzyl alcohol, phenolic compounds such as phenol, or quaternarycompounds such as benzalkonium chloride.

Any pharmaceutical used for therapeutic administration can be sterile.Sterility is readily accomplished by for example filtration throughsterile filtration membranes (e.g., 0.2 micron membranes). Anypharmaceutically acceptable sterility method can be used in thecompositions of the invention.

The pharmaceutical composition comprising a first substance capable ofincreasing cAMP levels, and optionally, a second substance capable ofincreasing intracellular calcium levels, or derivatives or saltsthereof, will be formulated and dosed in a fashion consistent with goodmedical practice, taking into account the clinical condition of theindividual patient, the method of administration, the scheduling ofadministration, and other factors known to practitioners.

EXAMPLE

A pharmaceutical composition comprising ibudilast and bumetanide wasadministered to patients with a diagnosis of autism spectrum disorder asdefined in the DSM-5, or PDD-NOS or Asperger syndrome as defined in theDSM-IV.

Eligibility criteria: male sex, age 5-50, no current chronic illness, nohistory of active seizures within 1 year, normal liver, renal andthyroid function. Concomitant medications were permitted if doses werestable for at least 60 days prior to the initiation of the challengetest. Patients with no syndromic or identified genetic etiology.

Individuals with idiopathic ASD were classified as Phenotype 1 if theyshowed:

-   -   at least 1 mandatory characteristic:        -   enlarged head size versus control population characterized            by at least one standard deviations above the mean head            circumference at 24 months and/or formal macrocephaly            (HC>97% of the general population)            -   and/or        -   cyclical aggravation of core or ancillary autism symptoms            potentiated by periods of infectious events, deciduous tooth            loss, post-traumatic injury, endogenous and exogenous            temperature variation            -   and    -   at least 2, and most preferably at least 3 of the following 20        characteristics:        -   accelerated hair and nail growth versus control population,        -   increased tendency to present with lighter colors of skin            and eyes as compared to individuals of the same ethnicity,        -   substantially longer eyelashes than control subjects of the            same ethnicity,        -   at least 5 non-contiguous areas of hypopigmented skin,            particularly presenting on the back of the patient,        -   signs of edema during periods of infectious events,            deciduous tooth loss, post-traumatic injury, or endogenous            and exogenous factors modifying body temperature; more            specifically, facial edema located in the periorbital and            forehead areas,        -   increased blood levels of gamma-glutamyl transpeptidase            (GGT) as compared to typically developing individuals of the            same age and ethnicity,        -   congenital genitourinary malformations and/or functional            impairment to initiate urinating,        -   hypoplasia of the patella,        -   frequent episodes of diarrhea specifically before the age of            5 years,        -   frequent episodes of tinnitus,        -   thinning of the corpus callosum,        -   positive family history for hematological malignancies in            particular but not limited to myeloma and acute            promyelocytic leukemia,        -   positive family history for rheumatoid arthritis, that is at            least two affected first-degree relatives in two consecutive            generations,        -   adverse events in response to acetyl-salicylic acid or its            derivatives,        -   iris coloboma, either monolateral or bilateral,        -   sleep hyperhidrosis particularly in newborns, toddlers and            young children (notably increased night sweating during            infancy and childhood—often reported by relatives to            requires bed linen changes),        -   increased Th1/Th2 ratio (i.e. elevated levels of Interleukin            1 beta, Interleukin 6, TNF-alpha, Interferon gamma),        -   congenital accessory or duplicated spleen,        -   congenital absence of the cisterna chili,        -   reported history of mother suffering from viral or bacterial            infection during pregnancy and/or biologically confirmed            Maternal immune activation during pregnancy.

First Intervention

Description: Four of the patients characterized as above wereadministered ibudilast. The four subjects had varying levels offunctioning ranging from mild to severe, and varying different levels ofverbal and intellectual abilities. IQ values ranged from 61 to 86. Allwere males.

TABLE 1 Patient status prior to intervention ADI baseline score Patient1 Patient 2 Patient 3 Patient 4 ADI-r SI 28 20 14 27 ADI-r C 24 19 18 17ADI-r RI 7 5 7 7Four patients were treated with ibudilast at a daily total dosage of 0.6mg/kg, TID.

TABLE 2 Patient status after treatment for four weeks of treatment withibudilast. ADI baseline score Patient 1 Patient 2 Patient 3 Patient 4ADI-r SI 21 16 12 22 ADI-r C 20 15 14 14 ADI-r RI 6 5 5 7

After between two and four months of mono-treatment with ibudilast, lossof treatment efficacy was reported in all four patients with gradualreturn to pre-treatment baseline scores.

A new treatment regimen was introduced, this time ibudilast at a dailytotal dosage of 0.6 mg/kg, TID, was administered in conjunction withbumetanide at a daily total dosage of 0.08 mg/kg, BID (with a maximumupper daily dose of 2 mg/kg/day).

Following a mean length of administration of 8 days of bi-therapytreatment effect on reduction of ADI-r scores reflected restoration ofinitial monotherapy efficacy.

TABLE 3 Patient status after treatment for 8 days with ibudilast +bumetanide ADI baseline score Patient 1 Patient 2 Patient 3 Patient 4ADI-r SI 20 15 11 22 ADI-r C 20 13 14 14 ADI-r RI 7 5 6 5

What is claimed is:
 1. A pharmaceutical composition comprising a firstsubstance capable of raising intracellular cAMP levels, a secondsubstance capable of modulating intracellular calcium concentration, anda pharmaceutically acceptable carrier.
 2. The pharmaceutical compositionaccording to claim 1, wherein the substance capable of raisingintracellular cAMP levels is a PDE inhibitor.
 3. The pharmaceuticalcomposition according to claim 2, wherein the PDE inhibitor is a PDE4inhibitor or a PDE4-3 dual inhibitor.
 4. The pharmaceutical compositionaccording to claim 2, wherein the PDE inhibitor is selected from thegroup consisting of theophylline, enprofylline, pentoxifylline,dyphylline, caffeine, dipyridamole, roflumilast, crisaborole,apremilast, cilomilast, tetomilast, rolipram, (S)-rolipram,(R)-rolipram, amrinone, milrinone, enoximone, daxalipram (R-mesopram),lirimilast, AWD-12-281, cipamfylline, oglemilast, tofimilast, CI-1044,HT-0712, MK-0873, arofylline, CI-1018, T-2585, YM-976, V-11294A,piclamilast, atizoram, filaminast, SCH 351591, IC-485, D-4418, CDP-840,and L-826,141.
 5. The pharmaceutical composition according to claim 1,wherein the substance capable of raising intracellular cAMP levels is asubstance capable of activating a G protein-coupled adenosine A_(2A)receptor.
 6. The pharmaceutical composition according to claim 5,wherein the substance capable of activating a G protein-coupledadenosine A_(2A) receptor is selected from the group consisting ofregadenoson, bidenoson, adenosine, 2-phenilaminoadenosine,2-amino-4-(4-hydroxyphenyl)-6-[(1H-imidazol-2-ylmethyl)thio]-3,5-pyridinecarbonitrile,4-[2-[(6-amino-9-b-D-ribofuranosyl-9H-purin-2-yl)thio]ethyl]benzenesulfonicacid ammonium salt, 2-hexynyl-5′-N-ethylcarboxamidoadenosine, CGS-21680,and UK-432,097.
 7. The pharmaceutical composition according to claim 1,wherein the substance capable of modulating intracellular calciumconcentration is a retinoic acid-related orphan receptor-alpha (RORA)agonist.
 8. The pharmaceutical composition according to claim 1, whereinthe substance capable of modulating intracellular calcium concentrationis a calcium channel inhibitor or an inhibitor of the solute carrierfamily 12 member 1, solute carrier family 12 member 1, 2, 4 or solutecarrier family 12 member 1, 2, 4,
 5. 9. The pharmaceutical compositionaccording to claim 1, wherein the substance capable of modulatingintracellular calcium concentration is selected from the groupconsisting of dihydropyridines, phenylakylamines, benzothiazepines,indolazines, aminoglycosides, and 4-substituted derivatives ofsulfamoylbenzoic acid.
 10. The pharmaceutical composition according toclaim 9, wherein the 4-substituted derivative of sulfamoylbenzoic acidis a derivative of 4-substituted-3-amino-5-sulfamoylbenzoic acid. 11.The pharmaceutical composition according to claim 10, wherein thederivative of 4-substituted-3-amino-5-sulfamoylbenzoic acid is selectedfrom the group consisting of bumetanide, AqB007, AqB011, PF-2178, BUM13,BUM5, bumepamine, and mixtures thereof.
 12. The pharmaceuticalcomposition according to claim 1, wherein the substance capable ofmodulating intracellular calcium concentration is selected from thegroup consisting of nifedipine, niludipine, nicardipine, nimodipine,NZ-10.5, amlodipine, felodipine, diperdipine, emopamil, devapamil,verapamil, diltiazem, flunarizine, fluspirilene pimozide, fantofarone,nicergoline, neomycin, gentamycin, kanamycin, cisapride, clopamide,cyproheptadine, loratadine, domperidone, fentanyl, alfentanil,sufentanil, flecainide, indoramin, isonepecotic acids, ketotifen,lobeline, loperamide, mepivacaine, methylphenidate, minoxidil, nipecoticacid, paroxetine, pempidine, penfluridol, perhexiline, pipecolics acid,bupivacaine, cyclohexemide, thalidomide, terfenadine, trihexyphenidyl,clevidipine, lomerazine, fostedil, anipamil, torasemide, chlorthalidone,etacrynic acid, furosemide, trichlormethiazide, hydroflumethiazide,methylclothiazide, bumetanide, ibutilde, mibefradil, dronedarone,amiodarone, nisoldipine, nitrendipine, nilvadipine, gabapentine, andambroxol hydrochloride.
 13. The pharmaceutical composition according toclaim 1, wherein the substance capable of raising intracellular cAMPlevels is ibudilast, and the substance capable of modulatingintracellular calcium concentration is bumetamide.
 14. A method oftreating a patient diagnosed with Fragile X syndrome comprisingadministering to a patient in need thereof an effective amount of afirst substance capable of raising intracellular cAMP levels and aneffective amount of a second substance capable of modulatingintracellular calcium concentration.
 15. A method of treating a patientdiagnosed with autism spectrum disorder (ASD) comprising administeringto a patient in need thereof an effective amount of a first substancecapable of raising intracellular cAMP levels, and an effective amount ofa second substance capable of modulating intracellular calciumconcentration.
 16. The method according to claim 15, wherein the patientexhibits characteristics consistent with being an ASD phenotype 1patient.
 17. The method according to claim 16, wherein the substancecapable of raising intracellular cAMP levels is a PDE inhibitor.
 18. Themethod according to claim 17, wherein the PDE inhibitor is a PDE4inhibitor or a PDE4-3 dual inhibitor.
 19. The method according to claim17, wherein the PDE inhibitor is ibudilast.
 20. The method according toclaim 19, wherein ibudilast is administered to the patient at a dailytotal dosage ranging from about 1-150 mg, preferably from about 10-80mg, and more preferably from about 15-50 mg, divided among one, two, orthree doses.
 21. The method according to claim 19, wherein ibudilast isadministered orally once, twice, or thrice daily to the patient using adosage form that comprises 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50mg ibudilast, or a pharmaceutically acceptable salt thereof.
 22. Amethod of treating a patient diagnosed with autism spectrum disorder(ASD), wherein the treatment comprises (i) determining whether thepatient suffers from ASD phenotype 1, and ii) administering atherapeutically effective amount of a pharmaceutical compositioncomprising a first substance capable of raising intracellular cAMPlevels, and a second substance capable of modulating intracellularcalcium concentration to the patient if the patient suffers from ASDphenotype 1; wherein determining whether the patient suffers from ASDphenotype 1 includes at least one step selected from a) subjecting thepatient to a challenge test with Nrf2-activator, b) verifying forclinical signs of heightened expression of proliferation-associatedpathways, c) verifying for upregulation of Nrf2, or d) verifying for lowlevels of protein kinase A; and wherein it is determined that thepatient suffers from ASD phenotype 1 if the patient exhibits at leastone characteristic selected from negative behavioral response in thechallenge test with a Nrf2 inducer, clinical signs of heightenedexpression of proliferation-associated pathways, upregulation of Nrf2,and low blood levels of protein kinase A.